Bi6721c Special Methods of Microorganisms Analysis I. - practical course

Faculty of Science
Spring 2008
Extent and Intensity
0/3/0. 3 credit(s). Type of Completion: z (credit).
Teacher(s)
doc. RNDr. Alena Španová, CSc. (seminar tutor)
doc. Ing. Bohuslav Rittich, CSc. (seminar tutor)
RNDr. Aleš Kovařík, CSc. (seminar tutor)
Guaranteed by
doc. Ing. Bohuslav Rittich, CSc.
Department of Experimental Biology – Biology Section – Faculty of Science
Contact Person: doc. Ing. Bohuslav Rittich, CSc.
Timetable
Thu 9:00–11:50 Bpb,02012
Prerequisites (in Czech)
NOW( Bi6721 Spec. Meth. Microorg. Anal. I. )
Course Enrolment Limitations
The course is also offered to the students of the fields other than those the course is directly associated with.
The capacity limit for the course is 12 student(s).
Current registration and enrolment status: enrolled: 0/12, only registered: 0/12, only registered with preference (fields directly associated with the programme): 0/12
fields of study / plans the course is directly associated with
Course objectives
I. Princips of work with DNA on model of plasmid DNA. Cultivation of bacterial cells E.coli JM109(pUC19). Microisolation of plasmid DNA pUC19. Preparation of agarose gel, gel electrophoresis of DNA, visualisation of DNA on gel, documentation. Purification of DNA. Elimination of RNA using LiCl and RNase A, proteins elimination using phenol extraction, precipitation of DNA by ethanol. Linearisation of plasmid DNA with restrictase EcoRI and estimation of restriction fragment lenght using program Anagel. II. PCR Salmonella. Isolation and purification of bacterial DNA Salmonella typhimurium LB5000 and Escherichia coli JM109. Spectrophotometric estimation of DNA concentration and purity.Preparation of PCR mixture for specific amplification of gene of Salmonella. Carry out of PCR and detection of PCR product. PCR with crude cell lysate. Immunomagnetic separation (IMS)of cells Salmonella from milk. IMS-cultivation of Salmonella cells. IMS-PCR of Salmonella cells.
Syllabus
  • 1. Safety of work in microbiological and molecular biotechnological laboratory. Pipeting of small volumes of solvents using pippetmans. Preparation of media for cultivation of bacteria. 2. Cultivation of bacterial cells E.coli JM109 and E. coli JM109(pUC19). Estimation of the presence of plasmids using cultivation of cells on plates with antibiotic. 3. Microisolation of plasmid DNA. 4. Preparation of 1% agarose gel. Gel electrophoresis of DNA. Visualisation of DNA and gel documentation. 5. Purification of DNA. Removing of RNA using LiCl and RNase A.Removing of proteins using phenol extraction. 6. Precipitation of DNA with ethanol.Checking of RNA removing and estimation of small concentrations of DNA using gel electrophoresis with standards containing known amount of DNA. 7. Linearisation of plasmid DNA with restrction nuclease EcoRI. Gel electrophoresis of cleaved DNA with DNA standards of known length. 8. Estimation of length of restriction fragment of DNA. The use of program Anagel. 9. Preparation of Salmonella typhimurium LB5000 and Escherichia coli JM109 cells for isolation of chromosomal DNA. Deproteination of DNA using phenol extraction. Precipitation of DNA using ethanol. 10.Spectrophotometric estimation of concentration and purity of DNA. The estimation of DNA integrity using agarose gel electrophoresis. 11.Preparation of PCR mixture for specific amplification of gene Salmonella using purified DNA as DNA matrix. Negative control.PCR reaction. 12.Detection of PCR product using agarose gel electrophoresis. The characterisation of PCR laboratory. 13.Preparation of crude cell lysate for PCR.Preparation of PCR mixture using crude cell lysate as DNA matrix. Pozitive and negative controls. Provodení PCR a detekce PCR produktu. 14.Imunomagnetic separation (IMS)of cells Salmonella. IMS-cultivation (IMS-CM) of cells Salmonella. IMS-polymerase chain reaction (IMS-PCR)with cells Salmonella. 15.Identifikation of Salmonella cells in unknown sample using PCR.
Literature
  • F.Sambrook and D.W. Russell Molecular Cloning. A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press. 2001
Assessment methods (in Czech)
Cvičení je ukončeno zápočtem
Language of instruction
Czech
Follow-Up Courses
Further comments (probably available only in Czech)
The course is taught annually.
Information on course enrolment limitations: Přednost mají posluchači specializace mikrobiologie.
The course is also listed under the following terms Spring 2008 - for the purpose of the accreditation, Spring 2011 - only for the accreditation, Spring 2003, Spring 2004, Spring 2005, Spring 2006, Spring 2007, Spring 2009, Spring 2010, Spring 2011, spring 2012 - acreditation, Spring 2014, Spring 2016, Spring 2017, spring 2018, Spring 2019, Spring 2020, Spring 2021, Spring 2022, Spring 2024, Spring 2025.
  • Enrolment Statistics (Spring 2008, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2008/Bi6721c